When a living organism substance such as DNA or protein is observed, it is a general practice to use a method of performing marking with a fluorescent dye, irradiating excitation light such as a laser, and observing generated fluorescent light. In recent years, as a technique that can measure fluorescent light at a molecular level, there is an observation method employing evanescent light (Non Patent Literature 1). When light having an angle equal to or larger than a fixed angle is made incident from a medium having a high refractive index to a medium having a low refractive index, the incident light does not diffuse to the medium having the low refractive index and total reflection occurs. At this point, a phenomenon in which the light slightly oozes out occurs on a surface on the low-refractive index medium side of a boundary surface. The oozing-out light is called evanescent light. The intensity of the evanescent light is attenuated exponentially further away from a refractive index boundary plane. Excitation light intensity is 1/e at a distance of about 150 nm from the refractive index boundary plane. Therefore, by fixing an observation sample to the boundary surface, it is possible to irradiate the excitation light only on the sample near the boundary surface. Background light deriving from a free phosphor, Raman scattering of water, or the like is suppressed and a high-contrast image is obtained.
Further, there is proposed DNA sequencing employing the technique of one-molecular fluorescence detection explained above (Non Patent Literature 2). Sample DNA fragments that should be analyzed are captured at random by one molecule at a time on a substrate surface and expanded by substantially one base at a time. This is measured by the fluorescence detection employing the evanescent light to determine a base sequence. Specifically, a single target molecule is fixed on a solution layer side on the refractive index boundary plane by using protein binding of biotin and avidin. The target DNA molecule is captured into mold DNA as a matrix of DNA polymerase. A DNA chain extension reaction can be stopped by the presence of a protecting group. A step of performing a DNA polymerase reaction using four kinds of dNTP derivatives (MdNTP) having marks that could be detected, a step of sensing the captured MdNTP with fluorescent light or the like, and a step of returning the MdNTP to an extendable state are set as one cycle. A base sequence of sample DNA is determined by repeating the cycle.
The evanescent light irradiation has an advantage that it is possible to observe a low-background and faint signal but, on the other hand, has a disadvantage that complicated and accurate optical adjustment (for strictly controlling an angle of incidence (AOI)) is necessary and indispensable. As means for solving the problem, the total internal reflection fluorescence (TIRF) microscope described in Patent Literature 1 includes the mechanism for keeping oozing depth of the evanescent light constant. Specifically, an angle of incidence of excitation light is automatically controlled on the basis of relation information between an angle of incidence of the excitation light and oozing depth of the evanescent light recorded in advance so that oozing depth designated by a user is obtained.
The total internal reflection fluorescence (TIRF) X-ray analysis device described in Patent Literature 2 includes the means for properly setting an angle of incidence of excitation light. Specifically, excitation X-ray is shone on a sample on a substrate, the intensity of a reflected X-ray reflected on the surface of the substrate is measured by a sensor, and an angle of incidence of the excitation X-ray is controlled on the basis of the intensity.    CITATION LIST    PATENT LITERATURE    PATENT LITERATURE 1: JP-A-2006-189741    PATENT LITERATURE 2: JP-A-03-246452    NON PATENT LITERATURE    NON PATENT LITERATURE 1: Nature 1995, Vol. 374, pp. 555-559.    NON PATENT LITERATURE 2: PNAS 2003, Vol. 100, pp. 3960-3964.